Mechanical stress activates neurites and somata of myenteric neurons

The particular location of myenteric neurons, sandwiched between the 2 muscle layers of the gut, implies that their somata and neurites undergo mechanical stress during gastrointestinal motility. Existence of mechanosensitive enteric neurons (MEN) is undoubted but many of their basic features remain to be studied. In this study, we used ultra-fast neuroimaging to record activity of primary cultured myenteric neurons of guinea pig and human intestine after von Frey hair evoked deformation of neurites and somata. Independent component analysis was applied to reconstruct neuronal morphology and follow neuronal signals. Of the cultured neurons 45% (114 out of 256, 30 guinea pigs) responded to neurite probing with a burst spike frequency of 13.4 Hz. Action potentials generated at the stimulation site invaded the soma and other neurites. Mechanosensitive sites were expressed across large areas of neurites. Many mechanosensitive neurites appeared to have afferent and efferent functions as those that responded to deformation also conducted spikes coming from the soma. Mechanosensitive neurites were also activated by nicotine application. This supported the concept of multifunctional MEN. 14% of the neurons (13 out of 96, 18 guinea pigs) responded to soma deformation with burst spike discharge of 17.9 Hz. Firing of MEN adapted rapidly (RAMEN), slowly (SAMEN) or ultra-slowly (USAMEN). The majority of MEN showed SAMEN behavior although significantly more RAMEN occurred after neurite probing. Cultured myenteric neurons from human intestine had similar properties. Compared to MEN, dorsal root ganglion neurons were activated by neurite but not by soma deformation with slow adaptation of firing. We demonstrated that MEN exhibit specific features very likely reflecting adaptation to their specialized functions in the gut

Pain in pancreatic ductal adenocarcinoma: A multidisciplinary, International guideline for optimized management

Abdominal pain is an important symptom in most patients with pancreatic ductal adenocarcinoma (PDAC). Adequate control of pain is often unsatisfactory due to limited treatment options and significant variation in local practice, emphasizing the need for a multidisciplinary approach. This review contends that improvement in the management of PDAC pain will result from a synthesis of best practice and evidence around the world in a multidisciplinary way. To improve clinical utility and evaluation, the evidence was rated according to the GRADE guidelines by a group of international experts. An algorithm is presented, which brings together all currently available treatment options. Pain is best treated early on with analgesics with most patients requiring opioids, but neurolytic procedures are often required later in the disease course. Celiac plexus neurolysis offers medium term relief in a substantial number of patients, but other procedures such as splanchnicectomy are also available. Palliative chemotherapy also provides pain relief as a collateral benefit. It is stressed that the assessment of pain must take into account the broader context of other physical and psychological symptoms. Adjunctive treatments for pain, depression and anxiety as well as radiotherapy, endoscopic therapy and neuromodulation may be required in selected patients. There are few comparative studies to help define which combination and order of these treatment options should be applied. New pain therapies are emerging and could for example target neural transmitters. However, until better methods are available, management of pain should be individualized in a multidisciplinary setting to ensure optimal care.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

Background!#!Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine.!##!Methods!#!We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/-physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival.!##!Results!#!We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves.!##!Conclusion!#!For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

<div><p>Background & aims</p><p>The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC).</p><p>Method</p><p>Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis.</p><p>Results</p><p>Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic <i>Bifidobacterium longum</i> NCC2705 (SERPIN_BL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants.</p><p>Conclusion</p><p>Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS.</p></div

Proteome analysis of mucosal biopsy supernatants.

<p>Proteome analysis revealed significantly different protein levels and pattern in biopsy supernatants from IBS, HC or UC. (A) 204 proteins exhibit different levels between the three groups (<i>p</i> < 0.05, Benjamini-Hochberg adjusted). Hierarchical clustering of z-transformed protein levels reveals striking differences between biopsy supernatants. (B) Principal component analysis of all 22 samples using the 204 proteins corroborates this finding. (C) The subset of the 8 proteins which were significantly upregulated in the IBS patients vs. healthy controls were used to construct predictive models using linear discriminant analysis (left panel). All combinations consisting of 2 to 5 proteins (p1-p5) were examined. The prediction accuracy of each is shown as a dot in the figure. The combination of the three proteases cathepsin L1 (CTSL1), proteasome subunit alpha type 4 (PSMA4), and elastase 3A (ELA3A) resulted in the highest value of prediction accuracy of 98%, marked with an arrow. Single ROC curves plotted for the three protein predictors CTSL1, PSMA4, and ELA3A are shown in the right panel. The area under the curve (AUC), as a measure of the discriminatory value of individual or combinatorial proteins, showed the highest value for the protein combination. Note, for none of the individual proteins the specificity to discriminate between the HC and the IBS groups was lower than 85% at a given sensitivity of 75%.</p

Differentially expressed proteases in the biopsy supernatants of patients with IBS, UC, and controls.

<p>Differentially expressed proteases in the biopsy supernatants of patients with IBS, UC, and controls.</p

Nerve activation evoked by mucosal biopsy supernatants.

<p>(A, left panel) Mucosal biopsy supernatants from patients with irritable bowel syndrome (14 IBS) and patients with ulcerative colitis in remission (12 UC), but not from healthy controls (7 HC), caused nerve activation as indicated by a significantly increased neuroindex (product of spike frequency and % of responding neurons) (Dunn´s method). (A, right panel) the broad spectrum serine protease inhibitor FUT-175 reduced the neuroindex evoked by UC supernatants (Wilcoxon signed rank test for paired data). (B) Spike discharge in human submucous neurons after pressure application of supernatants (duration indicated by the bars below the traces) from IBS (upper panel) and UC patients (lower panel) before and during incubation with the PAR1 receptor antagonist SCH79797 (10μM). While SCH79797 blocked spike discharge in response to IBS supernatants, it had no effect on spike discharge after application of UC supernatants (each symbol represent one patient sample). (C) Quantification of the neuronal activity evoked by biopsy supernatants. The PAR1 receptor antagonist SCH79797 significantly reduced the neuroindex evoked by IBS supernatants from 14 patients (upper panel) but had no influence on the neuronal activation evoked by UC supernatants from 12 patients (lower panel) (each symbol represent one supernatant; Wilcoxon signed rank test for paired data). Numbers in parentheses indicate number of tissues/ganglia/neurons studied.</p